Stuart Patton

The epithelial mucin, MUC1, of milk, mammary gland and other tissues

MUC1 is a mucin-type glycoprotein that is integrally disposed in the apical plasma membrane of the lactating epithelial cell and protrudes from the cell surface into the alveolar lumen where milk is stored. Envelopment of milk fat globules by this membrane accomplishes their secretion and conveys MUC1 into milk. The human form of this mucin has been detected in many other organs, tissues and body fluids. It projects from the cell surface as long filaments. In the human and a number of other species, MUC1 is polymorphic due to variable numbers of a tandemly repeated segment 20 amino acids in length. The individual codominantly expresses two alleles for the mucin so that differences in its size among individuals and between the two forms of an individual are observed. The tandem repeats are rich in serines and threonines which serve as O-glycosylation sites. Carbohydrate content of MUC1, as isolated from milk of human, bovine and guinea pig, is approximately 50%. The oligosaccharides carry substantial sialic acid at their termini and this accounts for two putative functions of this mucin, i.e., to keep ducts and lumens open by creating a strong negative charge on the surface of epithelial cells which would repel opposite sides of a vessel, and to bind certain pathogenic microorganisms. MUC1 is protease resistant (trypsin, chymotrypsin and pepsin) and large fragments of it can be found in the feces of some but not all breast-fed infants. MUC1 has a highly varied structure because of its polymorphism, qualitative and quantitative variations in its glycosylation between tissues, individuals and species, and differences due to divergence in the nucleotide sequences among species. Sequencing of the MUC1 gene for various species is showing promise of revealing unique evolutionary relationships and has already indicated conserved aspects of the molecule that may be functionally important. Among these are positions of serine, threonine and proline in the tandem repeats and a high degree of homology in the transmembrane and cytoplasmic segments of the molecule.

Glycoproteins of the Human Milk Fat Globule in the Protection of the Breast-Fed Infant against Infections

Nonimmunological components in human milk can protect breast-fed infants against infection by microorganisms. The structural and functional characteristics of four such components are discussed. The mucin inhibits binding of S-fimbriated Escherichia coli to bucal epithelial cells; lactadherin prevents symptomatic rotavirus-induced infection; glycoaminoglycans inhibit binding of human immunodeficiency virus gp120 to its host cell CD4 receptor, and oligosaccharides provide protection against several pathogens and their toxins.

Carotenoids of human colostrum

Colostrum, the initial postpartum secretion of the breast, ordinarily has a distinct yellow color due to carotenoids of its fat globules. This pigmentation progressively diminishes as milk production increases during the first week of lactation. Identity of these cartenoids was investigated by means of thin-layer chromatography, high performance liquid chromatography and spectral analysis. α- and β-carotene, lycopene and β-cryptoxanthin were revealed as major chromogens. A component corresponding to lutein and/or zeaxanthin was also detected by both chromatographic techniques. Extracts of 23 saponified colostrum samples from 10 donors revealed considerable variation in total carotenoid concentration (0.34–7.57 μg/ml of colostrum). Multiparous mothers had greater mean colostrum carotenoid concentrations than did the primiparae, 2.18±1.94 vs 1.14±1.32 μg/ml, respectively. Seven of the eight primiparous donors samples had little or no yellow color. These findings imply a difference in carotenoid transport by breasts that have lactated as compared to those that have not. The interrelation of carotenoids, lactation and breast cancer is discussed.

Morphometric evaluation of lipid droplet associations with secretory vesicles, mitochondria and other components in the lactating cell

SummaryThe size, cellular location, and identity of surface-associated components were determined for lipid droplets in lactating cells. Transmission electron-microscopic measurements were made involving 3801 droplets in approximately 211 cells from three rats and 1197 droplets in 66 cells from a mouse. For the purposes of droplet evaluation, cells were divided into seven locations ranging from basal to secreting positions. Droplets were also categorized with respect to contact with other droplets, basolateral plasma membrane, mitochondria, Golgi apparatus, secretory vesicles, and endoplasmic reticulum-cytoplasm (ERC). Data on droplet size showed that droplet growth occurs mainly in the secretory position, confirming previously published findings. Lipid droplets from mouse tissue, although somewhat smaller in size showed similar growth trends to those of the rat. Data on numbers of droplet contacts and percentages of droplet circumferences involved in associations with other cell components showed that the dominant interaction of lipid droplets was with the ERC. However, intimate association of droplets with mitochondria was noted in all cellular locations. In addition, nursed animals exhibited a greater proportion of droplet surface association with secretory vesicles and less in contact with mitochondria in comparison to those not nursed. The significance of these relationships to milk synthesis and secretion is discussed.

Structural, histochemical and biochemical observations on horse milk-fat-globule membranes and casein micelles.

SummaryHorse milk fat glubules (MFGs) and casein micelles were studied using freeze fracturing, freeze etching and thin-section electron microscopy, as well as lectin histochemistry, gel electrophoresis, and Western blotting. Horse MFGs were found to be relatively small, their average volume-surface diameter being about 2.75 μm. The MFG membrane is composed of three layers: an inner proteinaceous coat occasionally having a paracrystalline substructure, a unit membrane, and a prominent filamentous glycocalyx. The last is rich in glycoconjugates, as revealted by its binding of various lectins. In addition, the glycocalyx binds cationized ferritin, which indicates the presence of negative electric charges. Gel electrophoresis revealed the presence of high-molecular-weight glycoproteins in the MFG membrane of horse milk. Such glycoproteins are also present in human MFG membranes but are absent in the bovine MFGs. The casein micelles in horse milk are relatively large, their average volume-surface diameter being about 200 nm.

Secretion of prosaposin, a multifunctional protein, by breast cancer cells

Western blotting and immunodetection with three antibodies were used to probe conditioned media of breast cancer cells (MDA231, MDA435, MCF-7) for prosaposin, a lysosomal protein that occurs in milk. It was readily detected in media from these cells, and from that of an sv40-transformed mammary epithelial cell, HBL100, but not from medium of human neural tumor cells (SK-N-MC). In cultures of MCF-7 cells, the prosaposin pattern of secretion over time closely resembled that of procathepsin D, another lysosomal protein occurring in milk. Supplementing medium with 17beta-estradiol (0. 1-100 nM) dose dependently increased secretion of both proteins after 48 h without changes in cell viability. The influence of 17beta-estradiol on secretion could play a role in the trophic activity of prosaposin in cellular differentiation and cell death protection. In concert with other lysosomal proteins in the tumor environment, such as procathepsin D, prosaposin may be a factor in eliminating barriers to tumor metastasis by facilitating hydrolysis of membrane glycolipids. The number of milk proteins known to be secreted by breast cancer cells is growing. There is evidence that at least some of these may be secreted in an endocrine manner in the normal, non-lactating breast.

Differences between individuals in high-molecular weight glycoproteins from mammary epithelia of several species.

Milk fat globules are secreted by envelopment in plasma membrane of the lactating cell. SDS-gel electrophoresis of proteins from this membrane has revealed differences between milk donors in two mucin-like glycoproteins. One of these glycoproteins resolves in 3% acrylamide stacking gel and the other in 4% running gel. The proteins vary in number of bands (one or two) and band mobilities. This polymorphism arises, at least in part, from expression of hypervariable genes. In this study, gel electrophoretic evidence of similar polymorphism in glycoproteins from cow, chimpanzee, horse and human milks is presented. In distinction to the other species, the cow expressed only one of these proteins which was detected in the running gel at Mr 180,000 to 200,000. The electrophoresis pattern for this protein from six cows was highly varied with respect to number (one or two) and position of bands. Peanut agglutinin, wheat germ agglutinin and concanavalin A all were bound specifically by bands of the bovine glycoprotein. Binding of concanavalin A distinguishes the bovine protein from the two human glycoproteins. Further studies of species differences should help shed light on the evolution of these unique glycoproteins and their possible functions in mother and young.

The effect of maternal diets on the mean melting points of human milk fatty acids

Triacylglycerols (TAG) are synthesized in the rough endoplasmic reticulum of the secreting cells of the mammary gland. The TAG must be liquid at body temperature for formation of microdroplets in the cell, for fusion of these into droplets, and for secretion of the droplets as globules (1). The core of the globule is TAG, and most of these must be liquid. The liquidity of TAG is related to their structure and to the melting points (MP) of the fatty acids (FA). For example: the MP of 16:0 is 63°C; 18:1t, 44°C; and 18:1c, 16°C. The MP of 16:0–16:0–16:0 is 66°C, 16:0–16:0–18:1, 37°C, and 16:0– 18:1–18:1, 24°C (2). The diets of lactating women contain wide-ranging amounts of the major FA: 10:0, 12:0, 14:0, 16:0, 18:0, 18:1t, 18:1c, 18:2, and 18:3. Changes in diets alter the kinds and amounts of the FA, and as a result, the TAG in milk (3). It would appear that ingestion of large quantities of FA with MP above 38°C, which includes all of those listed above except 10:0, 18:1c, 18:2, and 18:3, would raise the MP of milk TAG. In addition, the production of 10:0–14:0, which are synthesized in the mammary gland, is stimulated by maternal diets high in carbohydrate, and this should increase the MP of milk TAG. However, the influence of changes in the maternal diet on the liquidity or MP of milk fat has not been reported. In this paper we provide the mean melting points (MMP) of the FA in milks from women who consumed a variety of diets. Holman et al. (4) developed a measure of liquidity or fluidity of membranes. They calculated the MMP of FA in plasma phospholipids. They found, for example, that the MMP was 14.8°C for normal American omnivores and 21.3°C for patients with multiple sclerosis. The authors briefly described their method for calculating MMP. The contribution of the molar fraction of the MP of each FA was determined. All increments were summed and the MMP calculated. One of us (RGJ) was told (Holman, R.T., personal communication) to add 100 to each FA-MP so as to avoid negative numbers. Dr. Frank D. Gunstone (personal communication) evaluated our method for calculating MMP and simplified the procedure. A description of the procedure follows: (i) Determine the mol% of each FA. We used only the major milk FA listed above. Inclusion of all FA had little effect. (ii) Multiply each mol% by the (MP + 100) of each FA to obtain MP fractions. See Reference 2 for MP. (iii) Sum the MP fractions and subtract 100 to obtain the MMP of the FA mixture. The contents of the major FA in milks from women on different diets and the MMP of their FA are shown in Table 1. The diets in the paper by Insull et al. (5) were given sequentially to a patient, and their effects on the FA profiles of her milk samples determined. The ingestion of 228 g of corn oil/d increased the 18:2 + 18:3 of her milk from 9.0 to 42.0%, while the MMP dropped markedly. Milks from the Nigerian (6,7), Tanganyikan (8), and Bedouin women (8) contained FA with MMP close to body temperature. Lauric (12:0) and 14:0 contents were high in these milks because their synthesis in the mammary gland is increased by the consumption of large quantities of dietary carbohydrates. The fat contents and volumes of milk were not reported so we do not know if those high MMP affected the secretion of milk fat. The MMP of the milks from mothers who ate diets high in 18:1t (9) were lower than those who did not. This may be caused by their lower contents of 14:0. The fat contents of the milks were; high, 18:1t, 4.51; and low, 4.14%. The low MMP in milks from the mothers on vegetarian diets (10) were caused by the relatively large amount of 18:2, 28.8%, therein. The milk fat contents were; vegetarian, 3.1 and omnivorian, 4.2%. These were not significantly different when tested by the Wilcoxon rank sum method. However, this large difference may be biologically important. These results illustrate the value of determining and reporting the fat contents of the milks when FA are analyzed. The milks from all mothers, including those on unusual diets, had MMP at or below body temperature, thus all the milk fats should be liquid. The maintenance of milk TAG liquidity regardless of the ingestion of large quantities of FA with high MP indicates the ability of the mammary gland to synthesize TAG with the required MP and to incorporate them into milk fat globules. Human milk TAG are unique in that about 70% of the 16:0 are esterified to the sn-2 position (3,11–13). Based on their study, Winter et al. (11) stated that the amounts of 16:0 and 18:0 in TAG of human milk fat appear to be modulated by nonrandom distributions of 18:1c and 18:2 or 8:0–12:0, so that the fat is liquid at body temperatures. The small quantities of trisaturated TAG in human milk (11) will be dissolved in the liquid TAG. The acyltransferase systems in the gland are able to combine the FA into TAG that will be liquid at

Incidence and characteristics of cell pieces on human milk fat globules

For biochemical and nutritional purposes, milk fat globules of a species are assumed to be more or less uniform. The incidence of organelle-containing cytoplasm (crescents) on human milk fat globules was determined by fluorescence microscopy employing acridine orange. The percentage of globules with crescents in 20 predominantly morning samples from 17 donors ranged from 1 to 38% with three-quarters falling between 3 and 8%. However, analysis of morning and night samples from eight donors showed a significant nocturnal rise in the proportion of globules with crescents. Values for night samples were, on average, twice those for morning samples. Crescents deteriorate in human milk. These changes begin in the breast, with marked disintegration occurring in milk during 36-h storage at 2 degrees C. Crescents were isolated from globules by churning and separated from the released membrane by selective centrifugation. SDS-polyacrylamide gel electrophoresis showed that crescents contribute bands which render the globule protein pattern more complex. The significance of crescents in globule secretion, globule membrane preparation, infant nutrition and as a source of mammary cell components is discussed.

Release of remnant plasma membrane from milk fat globules by Triton X-100.

The nonionic detergent, Triton X-100, was investigated as an agent for releasing plasma membrane from milk fat globules. The sedimentable material (50 000 X g, 1 h) derived by treating washed goat globules with the detergent (0.2%) was compared to membrane made by the classical globule churning procedure. Characterization included lipid and protein analyses, gel electrophoresis of peptide components, determination of enzymatic activities, and examination with the electron microscope. The results established that the detergent-releasing material is membrane with similarities to the product by churning. Evaluation of variables revealed that a detergent concentration of 0.1 to 0.2% and reaction temperature of 20-22 degrees C appear optimum with respect to membrane yield when a reaction time of 2 min is employed. At higher detergent concentrations or temperatures removal of phospholipid from the membrane was maximized. Triton X-100 was observed to release membrane from milk fat globules of the goat, human and cow, the latter with a minor procedural modification. The detergent based method is a convenient procedure for obtaining plasma membrane material in good yield for biochemical studies. It also should aid investigations of milk fat globule structure.